Killing fungi by boosting fluconazole

Now to a potential strategy to boost the fungus-killing power of the commonly-used drug fluconazole. Jessica Brown has been looking for chemicals that can sensitise fungi to the effects of this drug. She’s began with another agent that has the effect she wants but isn’t suitable for clinical use, and she’s used that to select for test fungi that can highlight other potential compounds. As she explained to Chris Smith, the strategy has also revealed some potential drug combination “no-nos” that can actually cut the effectiveness of fluconazole... 

Jessica - Our goal was to improve the treatment of fungal infections called cryptococcal meningitis. And this is when an infection caused by the fungus cryptococcus neoformans spreads from the initial site in the lungs and then disseminates to places such as the brain. And there are relatively few drugs, fewer than there are for antibiotics, because the cellular structure of the pathogen is very similar to our cellular structure. What functionally this means for cryptococcus is that the treatment itself takes about a year. Patients receive high doses of an oral drug called fluconazole. And the problem with fluconazole is that it is what's called a fungistatic drug. So it will inhibit fungal cell growth. That'll cause the cells to become static, but it won't kill the cells, which means that if they're a solid organ transplant patient, they basically are on these drugs for life.

Chris - So what can you do to improve things?

Jessica - So we wanted to improve this by finding drugs that increase the activity of fluconazole, such that it will better kill a fungus and we can ideally eventually shorten these treatment times. So what we've done is we've identified drugs that amplify the activity of fluconazole. And in our case at least one of the drug pairs we found convert fluconazole from static to cidal.

Chris - Do you know how it does it?

Jessica - So our best hypothesis about how these drugs work is that they target a pathway that interacts with the pathway targeted by fluconazole such that when you inhibit two pathways simultaneously, you're then able to kill the cell instead of just causing it to grow more slowly or stop growing.

Chris - So how did you go about, in the first instance, finding these agents that could do this? What was the approach?

Jessica - What we did is we took fluconazole and we took a second drug that is known to show this interaction with fluconazole that we want, the synergistic interaction where they amplify each other's activities. The problem with the second drug is that it's an immunosuppressant. So we can't really use this to treat patients, but we can use it as a training drug and use its properties to try to identify other drugs that show similar properties. So what we did is we treated fungal cells that were either what's called wild type, a standard healthy fungal cell, or carries a mutation in a subset of genes. We then grew those fungal cells in the presence of these two drugs and we identified a subset of mutants that showed a certain response to each of these two drugs. Now these two drugs have very different targets within the fungal cell, and so normally genes whose mutants would show a response to fluconazole would not show a response to the second drug. However, we noticed that with just a couple of mutants, they've responded to both the fluconazole and the second drug. And so we then used those mutants in a much larger screen to identify additional drugs that showed that same response.

Chris - So you don't know what the changes that's happened in these cells, you just know that by some mechanism there is something in these cells that means that in the presence of both fluconazole and the other agent, these cells will die. And so you can now use them as almost a marker. So if we come along with another drug, they're going to be exquisitely sensitive to another drug that will work in the same way as the other agent that wasn't suitable enabling you to find other agents that will potentiate the action of the fluconazole.

Jessica - Exactly. And what this means is that because we are looking for things that show a hypersensitivity for anything that potentiates fluconazole, we can then use this in a very large scale screen. We screened a library of molecules that were already approved by the FDA to treat other diseases. And the logic behind this was that because they're approved for other indications, they can be used off label by physicians who think that this is an appropriate form of treatment. And thus they don't require expensive clinical trials and can get into the clinic much more quickly. And what we found is that for our most promising combination, we did see a considerable present killing after 24 hours of treatment.

Chris - And out of interest, what is that promising compound that you've identified?

Jessica - So one of our favourites is a drug called dicyclomine and this inhibits G protein coupled receptors. It's what's called orally bioavailable, which means it can be taken as a pill. You don't need to be hospitalized to receive IV treatment. And it's normally used to treat irritable bowel syndrome, it relaxes muscles. But in fungi we find that it acts very strongly in combination with fluconazole. And when we infect mice with a fungus, let that infection progress until the mice have a brain infection, and then start treating with them with fluconazole plus dicyclomine we see an over twofold increase in the time to death for these mice compared to just treating with fluconazole alone.

Chris - Is that agent an easy bedfellow with the kinds of drugs that we will also be using in the sorts of patients that you're seeking to treat though? Because some drug-drug interactions preclude the use of some combinations that we would love to use, but they remain out of scope. So will it work in that way?

Jessica - So that we haven't tested yet. I haven't seen data suggesting that interactions for dicyclomine are a major problem. This is a very important question though, because part of what we found in this study is critical antagonistic interactions with fluconazole, where antagonistic interactions are those that decrease the efficacy of a drug of interest.

Chris - And that was going to be my next question, which is did you discover the reverse effect, which is some drugs which actually we hadn't realized are really very bad for the ability of fluconazole to poison fungi and therefore we ought to avoid them in our patient population at all costs?

Jessica - Yes, we did. And this is a particular problem for some of our patient populations. And so what we found was that a subset of very commonly used antibiotics decreases the efficacy of fluconazole very substantially. And since these antibiotics are those that are frequently used to treat staphylococcus infections, this could be a problem for our patient population.